1. Field of the Invention
The present invention relates to modulating the immune system. In particular, the invention relates to oligonucleotide-based compounds that selectively stimulate an immune response through binding to and activating Toll-Like Receptor 3 (TLR3), and their use, alone or in combination with other agents, for treating or preventing diseases wherein modulation of TLR3 activity would be beneficial.
2. Summary of the Related Art
Toll-like receptors (TLRs) are present on many cells of the immune system and have been shown to be involved in the innate immune response (Hornung, V. et al., (2002) J. Immunol. 168:4531-4537). TLRs are a key means by which mammals recognize and mount an immune response to foreign molecules and also provide a means by which the innate and adaptive immune responses are linked (Akira, S. et al. (2001) Nature Immunol. 2:675-680; Medzhitov, R. (2001) Nature Rev. Immunol. 1:135-145). In vertebrates, this family consists of at least 11 proteins called TLR1 to TLR11, which are known to recognize pathogen associated molecular patterns (PAMP) from bacteria, fungi, parasites, and viruses and induce an immune response mediated by a number of transcription factors.
Some TLRs are located on the cell surface to detect and initiate a response to extracellular pathogens and other TLRs are located inside the cell to detect and initiate a response to intracellular pathogens. Table 1 provides a representation of TLRs, the known agonists therefore and the cell types known to contain the TLR (Diebold, S. S. et al. (2004) Science 303:1529-1531; Liew, F. et al. (2005) Nature 5:446-458; Hemmi H et al. (2002) Nat Immunol 3:196-200; Jurk M et al., (2002) Nat Immunol 3:499; Lee J et al. (2003) Proc. Natl. Acad. Sci. USA 100:6646-6651); (Alexopoulou, L. (2001) Nature 413:732-738).
TABLE 1Cell Types ContainingTLR MoleculeAgonistReceptorCellSurface TLRs:TLR2bacterial lipopeptidesMonocytes/macrophagesMyeloid dendritic cellsMast cellsTLR4gram negative bacteriaMonocytes/macrophagesMyeloid dendritic cellsMast cellsIntestinal epitheliumTLR5motile bacteriaMonocyte/macrophagesDendritic cellsIntestinal epitheliumTLR6gram positive bacteriaMonocytes/macrophagesMast cellsB lymphocytesEndosomalTLRs:TLR3double stranded RNA virusesDendritic cellsB lymphocytesTLR7single stranded RNA viruses;Monocytes/macrophagesRNA-immunoglobulinPlasmacytoid dendriticcomplexescellsB lymphocytesTLR8single stranded RNA viruses;Monocytes/macrophagesRNA-immunoglobulinDendritic cellscomplexesMast cellsTLR9DNA containing unmethylatedMonocytes/macrophages“CpG” motifs; DNA-Plasmacytoid dendriticimmunoglobulin complexescellsB lymphocytes
The signal transduction pathway mediated by the interaction between a ligand and a TLR is shared among most members of the TLR family and involves a toll/IL-1 receptor (TIR domain), the myeloid differentiation marker 88 (MyD88), IL-1R-associated kinase (IRAK), interferon regulating factor (IRF), TNF-receptor-associated factor (TRAF), TGF3-activated kinase1, IκB kinases, IκB, and NF-κB (see for example: Akira, S. (2003) J. Biol. Chem. 278:38105 and Geller at al. (2008) Curr. Drug Dev. Tech. 5:29-38). More specifically, for TLRs 1, 2, 4, 5, 6, 7, 8, 9 and 11, this signaling cascade begins with a PAMP ligand interacting with and activating the membrane-bound TLR, which exists as a homo-dimer in the endosomal membrane or the cell surface. Following activation, the receptor undergoes a conformational change to allow recruitment of the TIR domain containing protein MyD88, which is an adapter protein that is common to all TLR signaling pathways except TLR3. MyD88 recruits IRAK4, which phosphorylates and activates IRAK1. The activated IRAK1 binds with TRAF6, which catalyzes the addition of polyubiquitin onto TRAF6. The addition of ubiquitin activates the TAK/TAB complex, which in turn phosphorylates IRFs, resulting in NF-kB release and transport to the nucleus. NF-kB in the nucleus induces the expression of proinflammatory genes (see for example, Trinchieri and Sher (2007) Nat. Rev. Immunol. 7:179-190).
TLR3 signaling occurs through a MyD88 independent pathway that begins with the TLR3 ligand interacting with and activating TLR3, which exists as a homo-dimer. Following activation, TLR3 undergoes a conformational change, allowing recruitment of a TIR-domain-containing adapter-inducing interferon-β (TRIF), which activates TANK-binding Kinase 1 (TBK1). TBK1 phosphorylates and activates IRF-3, resulting in the activation of interferons α and β and ultimately the generation of an inflammatory immune response (see for example: Miggin and O'Neill (2006) J. Leukoc. Biol. 80:220-226).
As a result of their involvement in regulating an inflammatory response, TLRs have been shown to play a role in the pathogenesis of many diseases, including autoimmunity, infectious disease and inflammation (Papadimitraki et al. (2007) J. Autoimmun. 29: 310-318; Sun et al. (2007) Inflam. Allergy Drug Targets 6:223-235; Diebold (2008) Adv. Drug Deliv. Rev. 60:813-823; Cook, D. N. et al. (2004) Nature Immunol. 5:975-979; Tse and Horner (2008) Semin. Immunopathol. 30:53-62; Tobias & Curtiss (2008) Semin. Immunopathol. 30:23-27; Ropert et al. (2008) Semin. Immunopathol. 30:41-51; Lee et al. (2008) Semin. Immunopathol. 30:3-9; Gao et al. (2008) Semin. Immunopathol. 30:29-40; Vijay-Kumar et al. (2008) Semin. Immunopathol. 30:11-21).
The selective localization of TLRs and the signaling generated therefrom, provides some insight into their role in the immune response. The immune response involves both an innate and an adaptive response based upon the subset of cells involved in the response. For example, the T helper (Th) cells involved in classical cell-mediated functions such as delayed-type hypersensitivity and activation of cytotoxic T lymphocytes (CTLs) are Th1 cells. This response is the body's innate response to antigen (e.g. viral infections, intracellular pathogens, and tumor cells), and results in a secretion of IFN-gamma and a concomitant activation of CTLs.
TLR3 is known to localize in endosomes inside the cell and recognizes nucleic acids (DNA and RNA) and small molecules such as nucleosides and nucleic acid metabolites. TLR3 has been shown to recognize and respond to double stranded RNA (dsRNA) viruses (Diebold, S. S., et al., (2004) Science 303:1529-1531). In addition, it has been shown that small interfering RNA (siRNA) molecules and non-targeted dsRNA molecules can non-specifically activate TLR3 (Alexopoulou et al. (2008) Nature 413:732-738). However, this non-specific activation of TLR3 was determined to be dependent on a MyD88 pathway, indicating that such dsRNA molecules have the potential to generate immune responses that are not specific to TLR3.
In addition to naturally existing and synthetic dsRNA ligands for TLR3, other synthetic oligonucleotide analogs have been shown to activate TLR3. The poly-inosinic acid poly-cytidylic acid complex (poly(I:C)), a synthetic double stranded RNA molecule that is designed to mimic viral dsRNA, is composed of a long strand of poly(I) annealed to a long strand of poly(C). Due to the need for long strands, poly(I:C) compounds are routinely synthesized using enzymatic processes. As a result of the enzymatic synthesis, the size of poly(I:C) compounds and preparations is known to vary between 0.2 kb and 8 kb. Poly(I:C) has been shown to induce interferon (Field et al. (1968) Proc. Natl. Acad. Sci. U.S.A. 61:340). Subsequent to this discovery, it was determined that poly(I:C) induces interferon through activation of TLR3 and, as compared to dsRNA molecules, poly(I:C) is preferentially recognized by TLR3 (Alexopoulou et al. (2001) Nature 413:732-738; Okahira et al. (2005) DNA Cell Biol. 24:614-623). The interferon inducing properties of poly(I:C) as well as its preferential binding to TLR3 make poly(I:C) a desirable molecule for use at inducing interferon in vivo. However, poly(I:C) exists as long strands of nucleic acids that have been shown to form undesirable helix-with-loop structures (Ichikawa et al. (1967) Bul. Chem. Soc. Japan 40:2272-2277) and to have toxic properties when administered in vivo (Absher and Stinebring (1969) Nature 223:1023; Lindsay et al. (1969) Nature 223:717; Adamson and Fabro (1969) Nature 223:718; Leonard et al. (1969) Nature 224:1023). Thus, the medical, therapeutic, and prophylactic use of poly(I:C) is limited.
Attempts have been made to modify the structure of poly(I:C) to retain its immune stimulatory properties while reducing its toxicity (WO2008109083). These compounds insert mismatches into the poly(I:C) strand by replacing cytosine with uracil at defined positions throughout the double stranded molecule. The compounds are referred to as poly(I:C12U). However, these compounds have had limited therapeutic success because their in vivo efficacy has been questioned and they have been rejected by the U.S.A. Food and Drug Administration.
Thus, it would be desirable to have a selective TLR3 agonist that retains the immune stimulatory activity and therapeutic activity of a poly(I:C) oligonucleotide without the undesired enzymatic synthesis, helix-with-loop structures, toxicity, and lack of efficacy of the currently available poly(I:C), poly(I:C12U), and dsRNA compounds.